title: Modification of p115RhoGEF Ser(330) regulates its RhoGEF activity.
abstract: p115RhoGEF is a member of a family of Rho-specific guanine nucleotide exchange factors that also contains a regulator of G protein signaling homology domain (RH-RhoGEFs) that serves as a link between Gα13 signaling and RhoA activation. While the mechanism of regulation of p115RhoGEF by Gα13 is becoming well-known, the role of other regulatory mechanisms, such as post-translational modification or autoinhibition, in mediating p115RhoGEF activity is less well-characterized. Here, putative phosphorylation sites on p115RhoGEF are identified and characterized. Mutation of Ser(330) leads to a decrease in serum response element-mediated transcription as well as decreased activation by Gα13 in vitro. Additionally, this study provides the first report of the binding kinetics between full-length p115RhoGEF and RhoA in its various nucleotide states and examines the binding kinetics of phospho-mutant p115RhoGEF to RhoA. These data, together with other recent reports on regulatory mechanisms of p115RhoGEF, suggest that this putative phosphorylation site serves as a means for initiation or relief of autoinhibition of p115RhoGEF, providing further insight into the regulation of its activity.
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, 835 S. Wolcott Avenue Room E403 (m/c 868), Chicago, IL 60612, USA. crchow@uic.edu
authors.forename: Christina R
authors.initials: CR
authors.lastname: Chow
authors.forename: Nobuchika
authors.initials: N
authors.lastname: Suzuki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kawamura
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
authors.forename: Tohru
authors.initials: T
authors.lastname: Kozasa
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D027602:Serum Response Element; D015398:Signal Transduction; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D012694:Serine; D008156:Luciferases; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020742:rhoA GTP-Binding Protein
keywords: ATRA; All-trans retinoic acid; DH; DMEM; Dbl homology; Dulbecco's modified Eagle's medium; FBS; Fetal bovine serum; GAP; GTPase activating protein; Galpha13; HPLC–MS/MS; High pressure/tandem mass spectrometry; LARG; LC/MS/MS; LPA; Leukemia-associated RhoGEF; Liquid chromatography/tandem mass spectrometry; Lysophosphatidic acid; Nα-p-tosyl-l-lysine phenylalanine chloromethyl ketone; Nα-tosyl-l-lysine chloromethyl ketone; PAR-1; PDZ; PH; PMA; Phorbol 12-myristate 13-acetate; Pleckstrin homology; Post-synaptic density protein, discs large protein, zonula occludens-1; Protease-activated receptor 1; RH-RhoGEF; RU; Regulator of G protein signaling homology domain-containing Rho guanine nucleotide exchange factor; Response units; RhoA; S1P; SPR; Sf9; Signal transduction; Sphingosine-1-phosphate; Spodoptera frugiperda; Surface plasmon resonance; TLCK; TPCK; p115RhoGEF